Phosphorylation of a Calmodulin (CaM)-dependent phosphoprotein phosphatase, calcineurin, (CN) by a CaM-dependent protein kinase modifies a specific serine residue. Dephosphorylation occurs by a mechanism in which the catalytic site of a modified polypeptide hydrolyses its own phosphoserine bond. This autodephosphorylation can be regulated by effectors of CN enzymatic activity. Activators of the reaction were cations of Mn, Ca, and Ni. Ca\CaM, which stimulates intermolecular phosphatase activity, inhibits autodephosphorylation presumably by binding to a primary sequence of CN juxtaposed with the phosphoserine. Certain inhibitors of CN activated autodephosphorylation such as phenothiazine drugs (trifluoperazine, and chlorpromazine) and metal ion chelators (EGTA, and EDTA). In contrast to the finding of others who found that phosphorylation inhibited the dephosphorylation of certain substrates, the phosphorylated enzyme dephosphorylated Inhibitor-1 at a more rapid rate than the dephosphorylated CN. A soluble enzymatic activity in brain exists that rapidly dephosphorylates phosphoCN. This activity is a Ca\CaM independent and inhibited by nM concentrations of okadaic acid which is a characteristic of phosphoprotein phosphatase 2A. The ability to dephosphorylate phosphoCN is however dependent on denaturation of phosphoCN and maintenance of a low level of sodium dodecyl sulfate in the reactions. These features suggest the phosphoserine residue exists in a constrained structure which requires conformation change for facile dephosphorylation to occur. Antibodies obtained from rabbits immunized with synthetic peptides composed of conserved sequences found in CN and other phosphoprotein phosphatases bound radiolabeled CN. Antibodies affinity purified on peptide-Sepharose columns were incubated with radiolabeled CN and adsorbed to protein-A Sepharose. Bound CN was eluted by denaturating conditions.